Project 3: Demethylation of HPV-associated head and neck cancer to trigger APOBEC synthetic lethality and enhance immune response

摘要 人乳头瘤病毒(HPV)相关的颈部鳞状细胞癌(HNSCC)呈上升趋势 HNSCC的比例。在过去的20年里，HPV+HNSCC的发病率急剧上升， 2012年超过宫颈癌成为美国最常见的HPV相关恶性肿瘤，尽管 HPV疫苗，估计到2060年，由HPV引起的HNSCC疫情不会减弱。 HPV+HNSCC发生在较年轻的个体中，与这些肿瘤患者的预后相比， 对于经典型HNSCC患者；然而，约25%的患者复发，几乎没有有效的治疗选择。 基于观察到的来自TCGA的HPV+HNSCC的超甲基化，以及对HPV使用 高甲基化以阻碍先天免疫反应，去甲基剂，5-氮杂胞苷(5- AzaC)，在HPV+HNSCC上进行检测。我们发现HPV+HNSCC细胞在培养和异种移植中 对5-azaC敏感，并且5-azaC引起双链断裂(DSB)，这在5-azaC之后没有观察到 治疗HPV阴性的HNSCC，即使剂量要高得多。我们发现，在5-azaC治疗后， 载脂蛋白B基因编码酶催化多肽3B(APOBEC3B)与 HPV+HNSCC中有染色质，而HPV阴性细胞中无染色质。CRISPR击倒A3B预防了DSB和 保护细胞免受5-azaC诱导的死亡。尽管DSB是必需的，但5- 未经治疗的HPV+HNSCC的克隆存活也需要azaC、A3B。这些数据表明，A3B 是HPV+HNSCC细胞生存所必需的，但随后的去甲基化A3B介导了毒性和DSB。 此外，5-azaC治疗增加了I型干扰素信号，这是通过增加 干扰素刺激的基因。这些令人兴奋的临床前数据导致了5-azaC为期5天的窗口试验。分析 的肿瘤标本证实了体外数据显示5-azaC导致了细胞毒性。免疫荧光 对一例HPV阳性患者在5-azaC治疗前后肿瘤的染色显示，与肿瘤相关的 淋巴细胞，可能是通过激活I型干扰素和增加 新抗原。在本研究中，我们假设5-azaC治疗将增强对nivolumab的反应。 (Nivo)通过其导致细胞死亡的能力，增加新抗原的表达，增加A3B驱动的突变 负载，并通过增加I型干扰素信号增强T细胞的渗透。这些假设将是 使用已建立的和新的体外试验以及通过治疗前后的检查进行测试 来自三臂临床试验的肿瘤标本。在目标1中，来自孢子窗试验的肿瘤标本将被 分析以确定5-azaC、Nivo或其组合对细胞死亡、细胞增殖、免疫的影响 渗透和免疫激活。Aim 2将使用标准和新的分析方法来探索A3B在 5-azaC治疗暴露的细胞毒性。在目标3中，我们将确定5-氮杂C对 尼沃存在或不存在时的免疫识别和反应。

SUMMARY Human papillomavirus (HPV)-associated neck squamous cell carcinoma (HNSCC) represents an increasing proportion of HNSCC. The incidence of HPV+ HNSCC has dramatically increased over the last 2 decades and in 2012 surpassed uterine cervical cancer as the most common HPV-related malignancy in the U.S. Despite the HPV vaccine, it is estimated that the “epidemic” of HNSCC caused by HPV will not diminish until 2060. HPV+ HNSCCs occur in younger individuals and prognosis for patients with these tumors is better compared to patients with classical HNSCC; however, ~25% of patients recur with few effective therapeutic options. Based on observed hypermethylation of HPV+ HNSCC from TCGA, and understanding that HPV uses hypermethylation to impede the innate immune response, effects of the demethylating agent, 5-azacytidine (5- azaC), were tested on HPV+ HNSCC. We found that HPV+ HNSCC cells in culture and xenografts are sensitive to 5-azaC, and that 5-azaC caused double strand breaks (DSB) that were not observed after 5-azaC therapy in HPV-negative HNSCC, even with much higher doses. We found that following 5-azaC therapy, APOlipoprotein B mRNA-Editing enzyme Catalytic polypeptide 3B (APOBEC3B) was associated with chromatin in HPV+ HNSCC, but not HPV-negative cells. CRISPR knockdown of A3B prevented DSB and protected cells from 5-azaC-induced death. Despite being required for DSBs and cellular toxicity caused by 5- azaC, A3B was also required for clonogenic survival of untreated HPV+ HNSCC. These data showing that A3B is required for survival of HPV+ HNSCC cells, but that following demethylation A3B mediates toxicity and DSB. In addition, 5-azaC therapy increased type I interferon signaling as measured by increased expression of interferon-stimulated genes. These exciting pre-clinical data led to a window trial of 5days of 5-azaC. Analysis of tumor specimens confirmed in vitro data showing that 5-azaC resulted in cellular toxicity. Immunofluorescent staining of an HPV+ patient tumors pre- and post-5-azaC showed a marked increase in tumor-associated lymphocytes, possibly driven through activation of type I interferon combined with increased expression of neoantigens. In this YHN-SPORE project, we hypothesize 5-azaC therapy will enhance response to nivolumab (Nivo) through its ability to cause cell death, increase neoantigen expression, increase A3B-driven mutational load, and enhance T cell infiltration through increased type I interferon signaling. These hypotheses will be tested using established and novel in vitro assays, as well as through examination of pre- and post-therapy tumor specimens from a 3-armed clinical trial. In Aim 1, tumor specimens from the SPORE window trial will be analyzed to determine effects of 5-azaC, Nivo, or the combination on cell death, cell proliferation, immune infiltration and immune activation. Aim 2 will employ standard and novel assays to explore the role of A3B in cellular toxicity exposed by 5-azaC therapy. In Aim 3, we will determine effects of 5-azaC on activators of immune recognition and response in the presence or absence of Nivo.